Materials and methods for detecting coronavirus

Abstract

The disclosure relates to test kits and methods for detecting the presence of Coronavirus polynucleotides in a biological sample.

What is claimed is: 
     
         1 . A kit for the detection Coronavirus polynucleotides in a biological sample comprising:
 a primer pair, wherein at least one primer in the primer pair comprises the sequence set forth in SEQ ID NO: 1, wherein the nucleotide set forth in SEQ ID NO: 1 is modified with a detectable label or an endonuclease enzyme cleavage site;   wherein the primer pair is capable of detecting Coronavirus polynucleotides, if present, in the sample by recombinase polymerase amplification (RPA).   
     
     
         2 . The kit of  claim 1 , further comprising a running buffer. 
     
     
         3 . The kit of  claim 1 , further comprising a test strip. 
     
     
         4 . The kit of  claim 1 , wherein the Coronavirus polynucleotide is a polynucleotide from SARS-CoV-2, CoV-229E, CoV-NL63, CoV-OC43, CoV-HKU1, SARS or MERS. 
     
     
         5 . The kit of  claim 1 , wherein Coronavirus polynucleotide is a polynucleotide from SARS-CoV-2. 
     
     
         6 . The kit of  claim 1 , wherein the endonuclease enzyme is endonuclease IV (Nfo). 
     
     
         7 . The kit of  claim 6 , wherein the primer pair comprises a first primer comprising the nucleotide sequence set forth in SEQ ID NO: 16. 
     
     
         8 . The kit of  claim 7 , wherein the primer pair optionally comprises a second primer comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 17. 
     
     
         9 . The kit of any one of  claim 1 , wherein the detectable label is biotin. 
     
     
         10 . The kit of  claim 9 , wherein the primer pair comprises a first primer that a biotinylated forward primer set forth in SEQ ID NO: 1 and a second primer is a reverse primer labeled with 6-carboxyfluorescein (FAM) set forth in SEQ ID NO: 6. 
     
     
         11 . The kit of  claim 1 , wherein the test strip comprises filter paper. 
     
     
         12 . The kit of  claim 11 , wherein the test strip comprises chitosan. 
     
     
         13 . A method for detecting Coronavirus polynucleotides in a biological sample comprising:
 (a) an amplifying step comprising adding the biological sample to a vessel containing a primer pair that is capable of amplifying Coronavirus polynucleotides, if present, in the biological sample, wherein at least one primer in the primer pair comprises the sequence set forth in SEQ ID NO: 1, wherein at least one of the primers of the primer pair is modified with a detectable label and/or an endonuclease enzyme cleavage site,   (b) combining the double-stranded amplified product containing single-stranded tail(s) with a running buffer, and incubating the mixture for a period of time in the vessel; and,   (c) a detecting step comprising wicking the mixture into a test strip and visually detecting the amplified product on the test strip.   
     
     
         14 . The method of  claim 13 , wherein a second primer in the primer pair is modified with a different label than a first primer in the primer pair. 
     
     
         15 . The method of  claim 13 , wherein the running buffer comprises magnesium acetate. 
     
     
         16 . The method of  claim 15 , wherein the running buffer further comprises sodium chloride.